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The neural crest is a distinct population of embryonic cells found only in craniates. Several genes, including BMP-4and the zinc-finger transcription factor Slug, have been shown to mediate neural crest development in both chickens and mice. We examined the function of Slug in the frog Xenopus laevisby injecting antisense RNA (ASR) targeted to the 3' UTR of Xenopus Slug (XSlug). Injection of XSlugASR inhibits neural crest-cell migration and leads to reduction or loss of many neural crest derivatives, including pigment cells and cranial cartilages. Moreover, regions injected with XSlug ASR show significantly elevated levels of cell death, and expanded regions of the BMP-4antagonist, Chordin. In addition to its important role in neural crest migration, XSlug may mediate the cell-death-signaling pathway and other significant developmental pathways in Xenopus laevis. Supported by NSF.

Derivation of most if not all of the ossified skull from the embryonic neural crest is an accepted axiom of contemporary developmental biology. Yet, this 'fact' is based on direct empirical results involving just one species of vertebrate, the domestic chicken. Anuran amphibians (frogs) pose a particularly difficult challenge in this regard: bone typically forms many weeks after hatching, and tracing individual cells over this lengthy interval is technically difficult. We used RNA encoding for green fluorescent protein (GFP) to label premigratory neural crest in embryos of Xenopus laevis, and assessed cranial derivatives both before and after metamorphosis. Results confirm the neural crest contribution to larval cranial cartilages demonstrated previously by using conventional vital stains, lineage markers, and ablation techniques. They also provide evidence for the neural crest derivation of a major paired skull bone, the frontoparietal; other, later-forming bones are currently under study. This is the first direct empirical demonstration of neural crest contribution to the ossified skull in any amphibian. Supported by NSF.

In the chicken embryo, neural crest cells contribute to the connective tissue components of cranial muscle. Furthermore, a 'cryptic segmentation' exists, in which mesodermal and neural crest components of a given muscle are derived from the same visceral arch. Larval anurans have a highly apomorphic head. Unique features include, for example, second (hyoid) arch muscles that both originate and insert on cartilages from the first (mandibular) arch. This makes anurans an excellent testing ground for the hypothesis that cryptic segmentation is a conserved vertebrate trait that persists even in clades that have undergone drastic evolutionary changes in cranial morphology. Vital dye labelling and ablation of selected regions of migrating neural crest cells in Bombina orientalis indicate that neural crest contributes to connective tissue components at the attachment sites of several cranial muscles. Crest ablation severely disrupts cranial muscle patterning. Preliminary data indicate that cryptic segmentation is conserved in frogs. Supported by NSF (to J.H.) and by the Wenner-Gren Center Foundations and the Swedish Institute (to L.O.).

Slug is a Zn-finger transcription factor expressed in neural-crest cells. Slugs function has been studied in both mouse and chicken, with conflicting results, e.g. slug appears to be critical for neural crest migration in the chicken, but not in the mouse. To study the role of slug in amphibians, we injected fertilized eggs of Xenopus laevis with antisense slug RNA. This caused reduced expression of both slug and the related transcription factor snail, as assessed by in situ hybridization. In addition to gastrulation defects, we found a distinct inhibition of neural crest-cell migration and the reduction or loss of many neural crest derivatives. The effects of antisense slug RNA were not uniform, however. While rostral cartilages were often severely affected, and in some cases missing altogether, posterior cartilages always appeared quite normal. This suggests that the differentiation of these more caudal neural crest derivatives is less dependent upon slug/snail activity. These results are similar to those found in chicken and indicate that slug and snail are critical for neural crest development in Xenopus. Supported by NSF (T.F.C, J.H.), and NIH (M.W.K.).

The plethodontid salamander genus Thorius comprises a clade of miniaturized salamanders endemic to southern Mexico. Though the genus was erected (by E. D. Cope) more than 130 years ago, for more than 70 years following its initial description Thorius was thought to include just a single species. Nine named forms were added by 1970, and subsequent morphological and molecular studies have continued to reveal additional species. At present, 23 valid species are recognized, yet at least 11 undescribed species await formal description. These species coexist with and replace one another in complex geographic and elevational patterns, which include numerous instances of sympatry involving two, three, and possibly four species. Phylogenetic analyses suggest surprising trends in morphological evolution associated with miniaturization, including frequent homoplasy involving character loss and gain. While such cryptic diversity was not anticipated by early students of Thorius, it is proving to be a common theme in contemporary studies of amphibian systematics. The total number of valid, named species has been increasing at a rate of nearly 2% or more per year for the last two decades.

Most anuran amphibians exhibit a complex, biphasic life history consisting of a free-living aquatic larva (tadpole) and a terrestrial adult. Metamorphosis in anurans is characterized by extensive remodeling of larval tissues via growth or resorption and the de novo formation of many adult structures. This process is dependent on thyroid hormone, which initiates--via nuclear receptors--a cascade of gene expression in responding tissues. It has been hypothesized that changes in the thyroid hormone-regulated metamorphic program mediate the evolutionary diversification of larval and adult morphology. This could be accomplished via temporal changes in tissue sensitivity and responsiveness to thyroid hormone on a tissue or organ-specific level. We are interested in the role that thyroid hormone response plays in the diversification of a specific tissue, Meckel's cartilage, a prominent skeletal element in the larval lower jaw. We investigated the presumed ancestral pattern of thyroid hormone receptor (TRa and TRa) expression during tadpole development and metamorphosis, and compared this to patterns in other species with varied jaw morphologies associated with larval feeding adaptations. Additionally, we assessed whether patterns of cell death and proliferation correlate with thyroid hormone response. This study will contribute to our understanding of the biphasic developmental program of anuran amphibians and its role in the generation of anuran diversity.